CN109371022A - A kind of circular rna hsa_circKPNA2_002 and its specificity amplification primer and application - Google Patents
A kind of circular rna hsa_circKPNA2_002 and its specificity amplification primer and application Download PDFInfo
- Publication number
- CN109371022A CN109371022A CN201811508139.1A CN201811508139A CN109371022A CN 109371022 A CN109371022 A CN 109371022A CN 201811508139 A CN201811508139 A CN 201811508139A CN 109371022 A CN109371022 A CN 109371022A
- Authority
- CN
- China
- Prior art keywords
- primer
- circular rna
- temperature
- amplification
- ddh
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/50—Physical structure
- C12N2310/53—Physical structure partially self-complementary or closed
- C12N2310/532—Closed or circular
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA
Abstract
The invention discloses a kind of circular rna hsa_circKPNA2_002 and its specificity amplification primer and applications, wherein, the nucleotide sequence of the circular rna is as shown in SEQ ID NO:1, and high expression, can be used as the molecular marker of screening colorectal cancer in Colorectal Carcinoma；The present invention also provides a kind of for expanding the specificity amplification primer of the circular rna, the specificity amplification primer includes upstream primer and downstream primer, wherein, the nucleotide sequence of the upstream primer is as shown in SEQ ID NO:2, and the nucleotide sequence of the downstream primer is as shown in SEQ ID NO:3；The invention also discloses application of the specificity amplification primer in the kit that preparation is used for colorectal cancer screening and the kits including the specificity amplification primer.
Description
Technical field
The invention belongs to oncomolecularbiology field more particularly to a kind of circular rnas for diagnosis of colorectal carcinoma, use
Specificity amplification primer and application in the circular rna.
Background technique
Colorectal cancer is the malignant tumour occurred in lower digestive tract, has become global public health problem.With
The raising of living standards of the people, the disease incidence of colorectal carcinoma are in significantly raised trend, disease incidence and lethality in recent years
It ranks among the best, seriously affects people's lives quality.Early diagnosis, early treatment can greatly reduce colorectal cancer deterioration
Further occurrence occurs with the development of molecular level and to colorectal cancer, development, treats further grinding for related mechanism
Study carefully, screening to targeting diagnosis and targeted therapy molecular marker and research become colorectal cancer study field of greatest concern it
One.
Circular rna (Circular RNA, circRNA) is a kind of without the end 5' cap and the end 3' poly (A) tail
Bar and with covalent bond formed ring structure non-coding RNA molecule, variable sheer is mainly passed through by precursor RNA (pre-mRNA)
Processing generates.Most of circRNA is positioned in cytoplasm, and minority is positioned in nucleus, most of to be formed by exon.
CircRNA is not easy to be degraded by exonuclease, can more stably be present in organism compared with linear rna, different
There is conservative in species, while there is expression specificity in different tissues and stage of development, most of circRNA are different
Between species rich content and all have tissue specificity, have stable expression in tissue, saliva, blood and excretion body,
This makes circRNA become the ideal biological marker that tumor patient diagnosis, treatment and prognosis are tracked.
In recent years, about the existing many reports of the relationship research between circRNA and tumour, clear circRNA has
Have and adjusts the functions such as tumor cell proliferation, apoptosis, vascularization, metabolism and drug resistance.Zhang etc. passed through to different lactation periods
Two groups of mouse breast tissue carry out the detection of circRNA chip, find circRNA in different lactation mouse breast tissue
Expression have very big difference, two periods detect the most of differences of circRNA type.Therefore, circRNA may be Colon and rectum
The novel targets of the accurate diagnosis and treatment of cancer, discovery circRNA marker relevant with screening colorectal cancer, have early prevention and treatment colorectal cancer
There are important meaning and value.
Summary of the invention
The application's is designed to provide the following aspects:
In a first aspect, the application provides a kind of circular rna, the nucleotide sequence of the circular rna such as SEQ ID NO:1 institute
Show, wherein two nucleotide of starting are cyclic binding site with two nucleotide of most end.
Full name of the circular rna in circ-RNA database circBank is hsa_circKPNA2_002, referred to as
CircKPNA2_002, the number in circBase database are hsa_circ_0045475.
Fig. 1 is the gene structure figure of the circular rna, as shown in Figure 1, the circular rna hsa_circKPNA2_002
Sequence 325bp, between 66033225-66036882 on No. 17 chromosome positive-sense strands of the mankind, by KPNA2 gene
The shearing of the exon area (exon) 2-4 is cyclized, and CDS indicates the circular rna from code area.F and R respectively represents gene spy
The position of specific primer, arrow represent the direction of primer initiation.
According to a second aspect of the present application, circular rna hsa_circKPNA2_002 described in first aspect present invention is provided
The purposes of molecular marker as screening colorectal cancer.
The research of the invention finds that relative to colorectal cancer patients Carcinoma side normal tissue, circular rna hsa_circKPNA2_
002 significantly raises in Colorectal Carcinoma.
Therefore, the circular rna hsa_circKPNA2_002 can be used for colorectal cancer screening.
Second aspect, the application also provide the purposes of molecular marker of the circular rna as screening colorectal cancer.
The third aspect, the application also provide it is a kind of for expanding the specificity amplification primer of circular rna described in first aspect,
The specificity amplification primer includes upstream primer and downstream primer, wherein
The nucleotide sequence of the upstream primer is as shown in SEQ ID NO:2；
The nucleotide sequence of the downstream primer is as shown in SEQ ID NO:3.
Specifically, the nucleotide sequence of the upstream primer are as follows: 5 '-GCTGCCAGCTTTCTCCCTTT-3 '；
The nucleotide sequence of the downstream primer are as follows: 5 '-GGGCAGCTGGTGTATTAGCA-3 '.
In a kind of achievable mode, the G/C content of the upstream primer is 55%, the G/C content of the downstream primer
It is 55%, wherein the G/C content refers in 4 kinds of bases of DNA, ratio shared by guanine and cytimidine.
Further, the TM value of the upstream primer is 60.9, the TM value 60.1 of the downstream primer, wherein the TM
Value refers to the melting temperature of upstream primer or downstream primer.
The inventors discovered that cancerous tissue and patient using specificity amplification primer provided by the present application to Patients With Rectal Carcinoma
RNA described in first aspect in itself cancer beside organism is expanded, and as shown in Examples 1 and 22, amplification is analyzed,
ROC curve is obtained, as a result as shown in figure 3, area is 0.840 under ROC curve, this shows to expand using specificity provided by the present application
Increase the amplified production that primer obtains as single band, no non-specific amplification, the specificity amplification primer can be used as this kind
The Specific marker of detection.
Fourth aspect, the application also provide a kind of method for expanding circular rna described in first aspect, which comprises
Step 1, synthesize the first chain cDNA: mixed raw material, the raw material include circular rna described in first aspect, with power traction
Object, ddH2O, dNTP mixed liquor, reverse transcription buffer, RNase inhibitor, reverse transcriptase are reacted according to the first temperature programmed control；
Step 2, PCR amplification is carried out to the first chain cDNA made from step 1: prepares amplification system, the amplification system packet
Include downstream primer, ddH shown in the cDNA of step 1 preparation, upstream primer, the third aspect shown in the third aspect2O, PCR amplification
Mix is reacted according to the second temperature programmed control.
In a kind of achievable mode, in step 1,
The mixing according to the following ratio of the raw material: circular rna described in 1 μ l first aspect, 1 μ l random primer, 10 μ l
ddH2O, 2 μ l dNTP mixed liquors, 4 μ l reverse transcription buffers, 1 μ l RNase inhibitor, 1 μ l reverse transcriptase, wherein described
DNTP mixed liquor includes dATP, dGTP, dCTP and dTTP；
First program are as follows: keep the temperature 5min under the conditions of 25 DEG C, then keep the temperature 60min under the conditions of 42 DEG C, finally 70
5min is kept the temperature under the conditions of DEG C.Further, reverse transcription is carried out using ABI PCR amplification instrument, it is standby that the cDNA is placed in -80 DEG C of storages
With.
Further, in step 2,
The amplification system is prepared according to the following ratio: the cDNA of 2 μ l steps 1 preparation, 0.8 μ l is as described in the third aspect
Upstream primer, downstream primer described in the 0.8 μ l third aspect, 6.4 μ l ddH2O, 10 μ l PCR amplification Mix；
Second program is the initial denaturation 30s at 95 DEG C, then to keep the temperature 5s at 95 DEG C, keep the temperature 20s at 61 DEG C
It is recycled at progress 40, then keeps the temperature 5s at 95 DEG C, 60s is kept the temperature at 60 DEG C, finally in 72 DEG C of reaction 5min.
5th aspect, the application also provide the kit that aforementioned specificity amplification primer is used for colorectal cancer screening in preparation
In application.
6th aspect, the application also provide a kind of kit for colorectal cancer screening, and the kit includes third
Specificity amplification primer described in aspect.
In a kind of achievable mode, the kit further includes reverse transcriptase, buffer, ddH2O, archaeal dna polymerase
At least one of with dNTP mixed liquor.
The hsa_ in the method detection Colorectal Carcinoma of real-time fluorescence quantitative PCR can be used in the kit
CircKPNA2_002 is used for screening colorectal cancer.
Applicants have discovered that high expression is presented in circular rna described herein in Colorectal Carcinoma cell, that is, knot
The content of circular rna described in rectum cancer tissue is much higher than the rna content in cancer beside organism, therefore, the circular rna hsa_
CircKPNA2_002 may be used as the molecular marker of colorectal cancer screening, moreover, provided by the present application for expanding the ring
The specificity amplification primer of shape RNA has high stability, specificity and sensibility to the circular rna, wherein in the tissue
Detection sensitivity up to 80%, specificity is up to 90%.
Detailed description of the invention
Fig. 1 shows hsa_circKPNA2_002 gene structure figure；
Fig. 2 shows real-time fluorescence quantitative PCR detection hsa_circKPNA2_002 in Colorectal Carcinoma and cancer beside organism
Differential expression figure；
Fig. 3 shows the ROC curve of hsa_circKPNA2_002 testing result in Colorectal Carcinoma.
Specific embodiment
The following describes the present invention in detail with reference to examples, the features and advantages of the invention will with these explanation and
It becomes more apparent from, is clear.But these examples are only exemplary, do not constitute any limit to protection scope of the present invention
System.
Embodiment
In embodiment, hsa_circKPNA2_002 screening the primer is limited by giving birth to work bioengineering (Shanghai) share
Company's synthesis；Trizol Reagent (article No. 15596018) is purchased from Invitrogen company；Chloroform (article No. 20100927) purchase
From Beijing chemical industry, reverse transcription reagent box is purchased from Thermo company (article No. K1622)；Real time fluorescent quantitative SYBR Premix Ex
Taq II (article No. RR820) is purchased from Takara.
Clinical sample: the cancerous tissues of 10 Patients with Colorectal Cancer, cancer beside organism's sample are by institute of hospital general of Ningxia Medical University
It provides.
Expression of the 1 PCR reaction detection hsa_circKPNA2_002 of embodiment in Colorectal Carcinoma
1, specificity amplification primer is designed:
Specificity amplification primer sequence and relevant information designed by the applicant are as shown in table 1:
1 specificity amplification primer sequence of table and relevant information
2, the extraction of total serum IgE:
(1) Trizol method extract Colorectal Carcinoma in total serum IgE: take 1g Colorectal Carcinoma, under the conditions of liquid nitrogen into
1mL Trizol is added in liquid nitrogen and continues to grind, to which Trizol to be ground to until the tissue is in pulverulence for row grinding
It after dry powder, is stored at room temperature, the centrifugation that all liq is transferred to 1.5ml RNase-free is collected after dry powder turns to liquid
Guan Zhong；
(2) fluid sample made from step 1 cracks after five minutes at room temperature, adds according to the Trizol grinding homogenate of every 1mL
Chloroform is added in the ratio for entering 0.2mL chloroform, covers tightly pipe lid, is vortexed after concussion 15s, stands 5min, under the conditions of 4 DEG C 12000rpm from
The heart 15 minutes, mixing liquid was layered as lower layer's chloroform phase after centrifugation, and middle layer albumin layer, upper layer colourless aqueous phase, RNA is whole to be assigned
In water phase；
(3) water phase is transferred in new centrifuge tube, 0.5mL isopropanol is added according to the Trizol grinding homogenate of every 1mL
Ratio isopropanol is added into system, mix, after -20 DEG C of precipitating 30min, 12000rpm centrifugation 15min, is obtained under the conditions of 4 DEG C
Precipitating, RNA are all present in precipitating；
(4) supernatant is removed, pre-cooling is added into system according to the ratio that 1mL is added in every 1mL Trizol into system
75% ethyl alcohol cleans RNA precipitate 2 times, mildly vibrates centrifuge tube, and suspend precipitating, and 7500rpm is centrifuged 5 minutes under the conditions of 4 DEG C；
(5) ethanol solution is removed, is dried 5-10 minutes at room temperature, until ethyl alcohol volatilizees, but is completely dried RNA not, Xiang Ti
The ddH without RNA enzyme is added in system2Centrifuge tube is added in O water (DEPC water), and 60 DEG C are incubated for 5 minutes, obtains total serum IgE；
(6) total serum IgE extracted determines the concentration of total serum IgE using the concentration and purity of NanoDrop ND-2000 measurement RNA
It can satisfy the requirement of subsequent experimental with purity, total rna solution packing is placed in -80 DEG C of preservations.
3, the synthesis of the first chain cDNA sequence:
PCR pipe is taken to configure reverse transcription system, reverse transcription system are as follows: 1 μ L total serum IgE (about 500ng), 1 μ L random primer
(Thermo company), 10 μ L ddH2O (Tiangeng biotech firm), 2 μ L dNTP mixed liquors (dATP, dGTP, dCTP and dTTP,
Invitrogen company), 4 μ L reverse transcription buffers (Thermo company), 1 μ L RNase inhibitor (Thermo company), 1 μ L is anti-
Transcriptase (Thermo company), 20 μ L of total volume.
Reaction condition are as follows: keep the temperature 5min at 25 DEG C；60min is kept the temperature at 42 DEG C, keeps the temperature 5min at 70 DEG C.Use Veriti
96 well Thermal Cycler PCR amplification instruments carry out reverse transcription.The cDNA that reverse transcription obtains is placed in -80 DEG C long-term storage
It deposits or is saved backup in -20 DEG C.
4, hsa_circKPNA2_002 gene cDNA amplification verifying:
The cDNA for taking 2 μ L step 3 reverse transcriptions to obtain prepares system and carries out PCR amplification；
PCR amplification system are as follows: 2 μ L cDNA, upstream primer shown in 0.5 μ L table 1, downstream primer shown in 0.5 μ L table 1,
10 μ L PCR amplification Mix, 7 μ L ddH2O, 20 μ L of total volume；
Reaction condition be 95 DEG C at initial denaturation 30s, then at 95 DEG C keep the temperature 5s, at 61 DEG C keep the temperature 20s and into
Row 40 circulations, then 5s is kept the temperature at 95 DEG C, 60s is kept the temperature at 60 DEG C, finally in 72 DEG C of reaction 5min.
Take 2 μ L reaction products in 1.5g agarose/100ml 1 × TAE buffer, 120V voltage is verified under the conditions of 25min
Whether PCR product is single specificity amplified band.
5, pcr amplification reaction:
The cDNA sample obtained using reverse transcription is detected.
Reaction system are as follows: 2 μ L cDNA, 0.8 μ L upstream primer, 0.8 μ L downstream primer, 6.4 μ L ddH2O (Tiangeng biology
Company), 10 μ L fluorescent quantitative PCR Mix (SYBGREEN dyestuff), 20 μ L of total volume；
Real-time PCR reactions condition: 95 DEG C of initial denaturation 5min；Then to keep the temperature 10s at 98 DEG C, heat preservation 30s is carried out at 62 DEG C
40 circulations.
Amplified reaction carries out on real-time fluorescence quantitative PCR instrument LightCyler480, expands while amplifying target genes
Increase the control of GAPDH internal reference, the primer gene order table such as SEQ ID NO:4 (upstream primer) and SEQ ID of the internal reference control
Shown in NO:5 (downstream primer), and pass through 2(-△△CT)The relative expression quantity of method calculating gene.
For embodiment 1, different samples are taken respectively, are repeated 10 times.
Wherein, as can be seen that amplified peak list in hsa_circ_0045475 gene real-time fluorescence quantitative PCR solubility curve figure
One, no miscellaneous peak, it was demonstrated that primer specificity is outstanding, and amplification experiment is normal.
Expression of the 2 PCR reaction detection hsa_circKPNA2_002 of embodiment in colorectal cancer cancer beside organism
The process of embodiment 1 is repeated, difference is: in step 2, extracting the total serum IgE in colorectal cancer cancer beside organism.
For embodiment 2, different samples are taken respectively, are repeated 10 times.
The PCR interpretation of result of Examples 1 to 2
1, gene expression analysis
Wherein, the expression of results of hsa_circKPNA2_002 is as shown in Figure 2:
In 10 pairs of tissues of Fig. 2, relative to colorectal cancer cancer beside organism, circular rna hsa_circKPNA2_002 exists
Expression in Colorectal Cancer significantly raises, 5 times of up-regulation or more.
The above result shows that circular rna hsa_circKPNA2_002 has in Colorectal Carcinoma and cancer beside organism
Differential expression, specifically, the circular rna hsa_circKPNA2_002 are low expression in Colorectal Carcinoma, therefore, described
Circular rna hsa_circ_0045475 can be used for the screening of colorectal cancer.
2, ROC curve is analyzed
The test data obtained to embodiment 1 and embodiment 2 is analyzed, and obtains ROC curve, as shown in Figure 3, wherein
Area under the curve AUC is 0.840, indicates that detection target spot can be as the Specific marker for detecting the circular rna, also, examine
Sensitivity is surveyed up to 80%, specificity is up to 90%.
One skilled in the art will appreciate that area is between 1.0 and 0.5 under ROC curve, and in the case where AUC > 0.5, AUC
Closer to 1, illustrate that diagnosis effect is better.AUC has lower accuracy in 0.5-0.7, and AUC is fixed in 0.7-0.9
True property, AUC have high accuracy at 0.9 or more, which, which is greater than 0.7, indicates the spy that detection target spot can be detected as this kind
Anisotropic marker.
Combine detailed description and exemplary example that the application is described in detail above, but these explanations are simultaneously
It should not be understood as the limitation to the application.It will be appreciated by those skilled in the art that without departing from the application spirit and scope,
A variety of equivalent substitution, modification or improvements can be carried out to technical scheme and embodiments thereof, these each fall within the application
In the range of.The protection scope of the application is determined by the appended claims.
Sequence table
<110>hospital general of Ningxia Medical University
<120>a kind of circular rna hsa_circKPNA2_002 and its specificity amplification primer and application
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 325
<212> DNA
<213>mankind (Homo sapiens)
<400> 1
ctttctccct ttgtctcata accatgtcca ccaacgagaa tgctaataca ccagctgccc 60
gtcttcacag attcaagaac aagggaaaag acagtacaga aatgaggcgt cgcagaatag 120
aggtcaatgt ggagctgagg aaagctaaga aggatgacca gatgctgaag aggagaaatg 180
taagctcatt tcctgatgat gctacttctc cgctgcagga aaaccgcaac aaccagggca 240
ctgtaaattg gtctgttgat gacattgtca aaggcataaa tagcagcaat gtggaaaatc 300
agctccaagc tactcaagct gccag 325
<210> 2
<211> 20
<212> DNA
<213>artificial sequence ()
<400> 2
gctgccagct ttctcccttt 20
<210> 3
<211> 20
<212> DNA
<213>artificial sequence ()
<400> 3
gggcagctgg tgtattagca 20
<210> 4
<211> 21
<212> DNA
<213>artificial sequence ()
<400> 4
tgttgccatc aatgacccct t 21
<210> 5
<211> 19
<212> DNA
<213>artificial sequence ()
<400> 5
ctccacgacg tactcagcg 19
Claims (9)
1. a kind of circular rna, which is characterized in that full name of the circular rna in circ-RNA database circBank be
Hsa_circKPNA2_002, nucleotide sequence is as shown in SEQ ID NO:1.
2. the purposes that circular rna described in claim 1 is used as the molecular marker of screening colorectal cancer.
3. the specificity amplification primer for expanding circular rna described in claim 1, which is characterized in that the specific amplification
Primer includes upstream primer and downstream primer, wherein
The nucleotide sequence of the upstream primer is as shown in SEQ ID NO:2；
The nucleotide sequence of the downstream primer is as shown in SEQ ID NO:3.
4. a kind of method of circular rna described in amplification claim 1, which is characterized in that the described method includes:
Step 1, synthesize the first chain cDNA: mixed raw material, the raw material include circular rna, random primer described in claim 1,
ddH2O, dNTP mixed liquor, reverse transcription buffer, RNase inhibitor, reverse transcriptase are reacted according to the first temperature programmed control；
Step 2, PCR amplification is carried out to the first chain cDNA made from step 1: prepares amplification system, the amplification system includes step
The cDNA of rapid 1 preparation, upstream primer as stated in claim 3, downstream primer as stated in claim 3, ddH2O, PCR expands
Increase mix, is reacted according to the second temperature programmed control.
5. according to the method described in claim 4, it is characterized in that, in step 1,
The mixing according to the following ratio of the raw material: circular rna described in 1 μ l claim 1,1 μ l random primer, 10 μ l ddH2O,
2 μ l dNTP mixed liquors, 4 μ l reverse transcription buffers, 1 μ l RNase inhibitor, 1 μ l reverse transcriptase, wherein the dNTP mixing
Liquid includes dATP, dGTP, dCTP and dTTP；
First program are as follows: keep the temperature 5min under the conditions of 25 DEG C, then keep the temperature 60min under the conditions of 42 DEG C, finally in 70 DEG C of items
5min is kept the temperature under part.
6. according to the method described in claim 4, it is characterized in that, in step 2,
The amplification system is prepared according to the following ratio: the cDNA of 2 μ l steps 1 preparation, on 0.8 μ l is as claimed in claim 3
Swim primer, 0.8 μ l downstream primer as claimed in claim 3,6.4 μ l ddH2O, 10 μ l PCR amplification Mix；
Second program be at 95 DEG C initial denaturation 30s, then at 95 DEG C keep the temperature 5s, at 61 DEG C keep the temperature 20s and
40 circulations are carried out, then keep the temperature 5s at 95 DEG C, 60s are kept the temperature at 60 DEG C, finally in 72 DEG C of reaction 5min.
7. application of the specificity amplification primer as claimed in claim 3 in the kit that preparation is used for colorectal cancer screening.
8. a kind of kit for colorectal cancer screening, which is characterized in that the kit includes spy as claimed in claim 3
Specific amplification primers.
9. kit according to claim 8, which is characterized in that the kit further include reverse transcriptase, buffer,
ddH2O, at least one of archaeal dna polymerase and dNTP mixed liquor.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811508139.1A CN109371022A (en) | 2018-12-11 | 2018-12-11 | A kind of circular rna hsa_circKPNA2_002 and its specificity amplification primer and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811508139.1A CN109371022A (en) | 2018-12-11 | 2018-12-11 | A kind of circular rna hsa_circKPNA2_002 and its specificity amplification primer and application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109371022A true CN109371022A (en) | 2019-02-22 |
Family
ID=65373276
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811508139.1A Pending CN109371022A (en) | 2018-12-11 | 2018-12-11 | A kind of circular rna hsa_circKPNA2_002 and its specificity amplification primer and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109371022A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110592220A (en) * | 2019-10-28 | 2019-12-20 | 郑州大学第一附属医院 | Early colorectal cancer diagnosis marker circ3823 and application thereof |
| CN110982906A (en) * | 2019-12-31 | 2020-04-10 | 广西医科大学 | Primer pair for detecting hsa _ circ _0032969, application thereof and kit |
| CN111088356A (en) * | 2019-12-31 | 2020-05-01 | 广西医科大学 | Primer pair for detecting hsa _ circ _0065793, application thereof and kit |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101932724A (en) * | 2007-10-05 | 2010-12-29 | 环太平洋生物技术有限公司 | The hyperplasia label and the prognosis of gastrointestinal cancer |
| US20120046186A1 (en) * | 2010-08-20 | 2012-02-23 | Pelham Robert J | Gene Expression Markers for Prediction of Response to Platinum-Based Chemotherapy Drugs |
| US20130064835A1 (en) * | 2009-10-08 | 2013-03-14 | Ann Marie Schmidt | Rage regulates rock activity in cardiovascular disease |
| CN107022627A (en) * | 2017-05-10 | 2017-08-08 | 哈尔滨医科大学 | The siRNA of application and the suppression KPNA2 gene expressions of KPNA2 genes application |
-
2018
- 2018-12-11 CN CN201811508139.1A patent/CN109371022A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101932724A (en) * | 2007-10-05 | 2010-12-29 | 环太平洋生物技术有限公司 | The hyperplasia label and the prognosis of gastrointestinal cancer |
| US20130064835A1 (en) * | 2009-10-08 | 2013-03-14 | Ann Marie Schmidt | Rage regulates rock activity in cardiovascular disease |
| US20120046186A1 (en) * | 2010-08-20 | 2012-02-23 | Pelham Robert J | Gene Expression Markers for Prediction of Response to Platinum-Based Chemotherapy Drugs |
| CN107022627A (en) * | 2017-05-10 | 2017-08-08 | 哈尔滨医科大学 | The siRNA of application and the suppression KPNA2 gene expressions of KPNA2 genes application |
Non-Patent Citations (2)
| Title |
|---|
| JULIA SALZMAN ET AL.: "hsa_circ_0045475", 《CIRCBASE》 * |
| 黄宏兴 等: "《骨质疏松实验研究概论》", 31 July 2018, 广东科技出版社 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110592220A (en) * | 2019-10-28 | 2019-12-20 | 郑州大学第一附属医院 | Early colorectal cancer diagnosis marker circ3823 and application thereof |
| CN110592220B (en) * | 2019-10-28 | 2023-04-14 | 郑州大学第一附属医院 | Early colorectal cancer diagnosis marker circ3823 and application thereof |
| CN110982906A (en) * | 2019-12-31 | 2020-04-10 | 广西医科大学 | Primer pair for detecting hsa _ circ _0032969, application thereof and kit |
| CN111088356A (en) * | 2019-12-31 | 2020-05-01 | 广西医科大学 | Primer pair for detecting hsa _ circ _0065793, application thereof and kit |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP3571322B9 (en) | Molecular subtyping, prognosis, and treatment of bladder cancer | |
| CN109371022A (en) | A kind of circular rna hsa_circKPNA2_002 and its specificity amplification primer and application | |
| CN107674916B (en) | Application of circular RNA in colorectal cancer biomarker | |
| CN105802964A (en) | cRNA-FUT8 detection applied to hepatocellular carcinoma screening and application thereof | |
| CN107519193A (en) | Esophageal squamous cell carcinoma early molecule diagnosis marker and its application | |
| CN108624693B (en) | MiR-577 is preparing the application in diagnosis of nephropathy marker | |
| CN108866187B (en) | Long-chain non-coding RNA marker related to lung cancer auxiliary diagnosis and application thereof | |
| CN107475441B (en) | Biomarker for predicting responsiveness of breast cancer patient to AT regimen neoadjuvant chemotherapy | |
| CN110268071A (en) | A kind of method and kit of the detection of PD-L1 expression | |
| CN111118146B (en) | Application of LncRNA in diagnosis of breast cancer chemotherapy-related myocardial injury patient | |
| CN109022583A (en) | Hsa_circ_0021977 is preparing the application on Diagnosis of Breast cancer product | |
| CN109371027A (en) | A kind of circular rna hsa-circ-0002938 and its specificity amplification primer and application | |
| CN109402123A (en) | A kind of circular rna hsa-circ-0073004 and its specificity amplification primer and application | |
| CN107058579A (en) | Adenocarcinoma of lung related miRNA, composition and its application | |
| CN106755309A (en) | Application of the molecular marked compound in cancer of pancreas prognosis evaluation product is prepared | |
| CN109280707A (en) | A kind of circular rna hsa_circPDE4D_040 and its specificity amplification primer and application | |
| CN109371023A (en) | A kind of circular rna hsa_circKIAA1199_006 and its specificity amplification primer and application | |
| CN109280706A (en) | A kind of circular rna hsa-circ-0102618 and its specificity amplification primer and application | |
| CN109402120A (en) | A kind of circular rna hsa-circ-0054020 and its specificity amplification primer and application | |
| CN109371024A (en) | A kind of circular rna hsa-circ-0073006 and its specificity amplification primer and application | |
| CN109161596B (en) | The application of miR-129 and its target gene in detection adenocarcinoma of lung | |
| CN109371029A (en) | A kind of circular rna hsa_circPDE4D_033 and its specificity amplification primer and application | |
| CN109371028A (en) | A kind of circular rna hsa_circPDE4D_044 and its specificity amplification primer and application | |
| CN109402119A (en) | A kind of circular rna hsa_circTGFBI_001 and its specificity amplification primer and application | |
| CN109402126A (en) | A kind of circular rna hsa-circ-0044513 and its specificity amplification primer and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20190222 |